Sulfation and amidinohydrolysis in the biosynthesis of giant linear polyenes

Clethramycin from Streptomyces malaysiensis DSM4137, and mediomycins (produced together with clethramycin from Streptomyces mediocidicus), are near-identical giant linear polyenes apparently constructed from, respectively, a 4-guanidinobutanoate or 4-aminobutanoate starter unit and 27 polyketide extender units, and bearing a specific O-sulfonate modification at the C-29 hydroxy group. We show here that mediomycins are actually biosynthesised not by use of a different starter unit but by direct late-stage deamidination of (desulfo)clethramycin. A gene (slf) encoding a candidate sulfotransferase has been located in both gene clusters. Deletion of this gene in DSM4137 led to accumulation of desulfoclethramycin only, instead of a mixture of desulfoclethramycin and clethramycin. The mediomycin gene cluster does not encode an amidinohydrolase, but when three candidate amidinohydrolase genes from elsewhere in the S. mediocidicus genome were individually expressed in Escherichia coli and assayed, only one of them (medi4948), located 670 kbp away from the mediomycin gene cluster on the chromosome, catalysed the removal of the amidino group from desulfoclethramycin. Subsequent cloning of medi4948 into DSM4137 caused mediomycins A and B to accumulate at the expense of clethramycin and desulfoclethramycin, respectively, a rare case where an essential biosynthetic gene is not co-located with other pathway genes. Clearly, both desulfoclethramycin and clethramycin are substrates for this amidinohydrolase. Also, purified recombinant sulfotransferase from DSM4137, in the presence of 3'-phosphoadenosine-5'-phosphosulfate as donor, efficiently converted mediomycin B to mediomycin A in vitro. Thus, in the final steps of mediomycin A biosynthesis deamidination and sulfotransfer can take place in either order.


Metabolite analysis and desulfoclethramycin isolation
For small-scale analysis, DSM4137 and S. mediocidicus were grown in liquid TSBY medium for 2-3 days. 1 mL samples of culture broth were centrifuged at 20,000g for 15 min.
The mycelia pellets were then extracted with 1 mL of methanol at 60 °C for 2 hours. The mixture was spun down and the clear methanol extract was evaporated to dryness and dissolved in 200 μL of methanol. 10 μL of the extract was analyzed by LC-UV-MS. LC-UV-MS analyses were performed on a HPLC (Agilent Technologies 1200 series) coupled to a Thermo Fisher LTQ mass spectrometer fitted with an electrospray ionization (ESI) source. The methanol extracts were loaded onto a Prodigy 5µ C18 column (4.6 × 250 mm, Phenomenex), and the samples were eluted using MQ containing 5 mM ammonium acetate (A) and acetonitrile (B) at a flow rate of 0.7 mL min −1 . The elution gradient for both extracts was 5% to 35% B over 10 min, 35% to 65% B over 30 min. The elution was monitored at 360 nm as well For desulfoclethramycin production and isolation, four 1 L Erlenmeyer flasks with spirals, containing 200 ml TSBY medium, were inoculated with 5 mL 2-day TSBY seed culture of S. malaysiensis DSM4137 and incubated at 30 °C, 200 rpm. After 2 days, the broth was centrifuged at 9,500 rpm for 30 min. The pellet was resuspended in methanol and incubated at 60 °C for 2 h. The suspension was centrifuged at 2500g for 10 min at room temperature and the supernatant, which showed a significant yellow colour, was transferred to a round bottom flask.
The solvent was evaporated and the water was removed by lyophilisation. The residue was dissolved in MeOH and purified by preparative HPLC (Agilent 1200) fitted with a Luna C18 column (100Å, 21.20 × 250 mm, Phenomenex). Compounds were eluted with 5 mM ammonium acetate (A) and MeOH (B) with a linear gradient of 5% to 60% B over 10 min, 60% B to 100% B over 20 min at a flow rate of 20 mL/min. Fractions were collected, and checked by MS analysis. Fractions containing desulfoclethramycin were combined. After removing methanol under reduced pressure, samples were lyophilized and kept at −20 °C before use.

Sulfotransferase gene knock-out in S. malaysiensis DSM4137
The knock-out of the sulfotransferase gene smala2697 in S. malaysiensis DSM4137 was performed by introducing an in-frame deletion. The construction of the deletion plasmid pYH7-smala2697 was achieved by i) PCR amplification of around 2 kbp DNA fragments upstream and downstream of smala2697, using pairs of primers smala2697-L1/L2 and smala2697-R1/R2, respectively, from genomic DNA of S. malaysiensis DSM4137; ii) NdeI restriction digestion of the cloning vector pYH7, followed by treatment with antarctic phosphatase AnP, and agarose gel purification; iii) ligation of the two fragments and the digested pYH7 plasmid by the isothermal assembly method as described previously [3], with a 50 °C for 60 min incubation step; iv) transformation of pYH7-slfV in E. coli DH10B; v) plasmid isolation, and PCR and sequencing confirmation of the inserted deletion fragment, using primers smala2697-CP1, smala2697-CP2, NdeI-L, and NdeI-R.
The pYH7-smala2697 construct was then introduced into S. malaysiensis DSM4137 by intergeneric conjugation. Freshly grown E. coli ET12567-pUZ8002-pYH7-smala2697 cultures at A 600 ~ 0.4-0.5 were thoroughly washed, to remove antibiotics, mixed with 2-3 days old Streptomyces mycelium, and plated on SFM agar. Following 20-22 h of incubation at 30 °C, plates were overlaid with nalidixic acid (25 μg mL −1 ) and apramycin (5 μg mL −1 ). Single Streptomyces colonies from these plates were streaked onto SFM agar containing 50 μg mL −1 apramycin, to confirm they had undergone antibiotic selection. Following further several rounds of incubation in a non-selective TSBY medium, mutants were screened for Apr S phenotype, by patching of single colonies onto both SFM agar and SFM agar containing apramycin (50 μg mL −1 ). To identify the mutants in which a double cross-over event had occurred, their genomic DNA was amplified with the smala2697-CP1/CP2 primer pair, and the resulting DNA fragments of the correct length (0.7 kb) were verified by sequencing.

Complementation of amidinohydrolases into S. malaysiensis DSM4137
The amidinohydrolase medi4948 was amplified by PCR, using as template genomic DNA of S. mediocidicus, and inserted into vector pIB139 via NdeI and EcoRV restriction sites to yield pIB139-medi4948.
The amidinohydrolase amh_A828 was amplified by PCR, using as template genomic DNA of Streptomyces olivaceus Tü4018, and inserted into vector pIB139 via NdeI and EcoRV restriction sites to yield pIB139-amh828.
The construct was then introduced by conjugation into S. malaysiensis DSM4137. The donor strain was E. coli ET12567/pUZ8002, and conjugation was carried out on 20 mL of SFM plates. After incubating at 30 °C for 20 hours, exconjugants were selected with 50 μg mL −1 apramycin and 25 μg mL −1 nalidixic acid. Single colonies from this plate were transferred to a SFM plate containing 50 μg mL −1 apramycin to double check for antibiotic resistance. The patch from the confirmation plate was then inoculated into TSBY liquid culture containing 50 μg mL −1 apramycin for production of metabolites.

Complementation of the sulfotransferase deletion mutant of S. malaysiensis DSM4137 using cloned slf genes
The in trans complementation of the S. malaysiensis DSM4137 sulfotransferase deletion mutant ∆smala2697 was done using the native smala2697, as well as sulfotransferase medi5536 from S. mediocidicus ATCC23936. Genes smala2697 and medi5536 were PCR amplified from genomic DNA, using primer pairs smala2697_com_F/R and medi5536_com_F/R, respectively. The cloning vector pIB139 was digested with NdeI and Eco321 and gel purified.
The smala2697 and medi5536 PCR fragments were ligated by the isothermal assembly method with the digested pIB139 plasmid, to yield plasmids pIB139-smala2697 and pIB139-medi5536, respectively. The latter plasmids were used to transform E. coli DH10B, the plasmids were isolated, and their identity confirmed by PCR and sequencing using primers S5 pIB-seqF and pIB-seqR. The constructs were then introduced by conjugation into the ∆smala2697 mutant. The conjugation procedure was as described in 1.4.

Protein expression and purification
The sulfotransferase gene smala2697 was amplified by PCR, using genomic DNA of S. malaysiensis DSM4137 as template, and inserted into vector pET28a via NdeI and HindIII restriction sites to yield pET28a-smala2697.
The three amidinohydrolase genes medi0234, medi2865 and medi4948 were individually amplified by PCR, using genomic DNA of S. mediocidicus, and inserted into vector pET28a via NdeI and HindIII restriction sites to yield pET28a-medi0234, pET28amedi2865, and pET28a-medi4948. The identities of the plasmids were confirmed by DNA sequencing.
The plasmids were then used to transform E. coli BL21(DE3) for protein expression.
A single colony was inoculated into 10 mL of LB medium containing 50 μg ml -1 kanamycin and grown overnight at 37 °C, 250 rpm. An aliquot (1 mL) was retained for preparation of a glycerol stock and the remaining culture was inoculated into 1 L LB medium containing 50 μg mL −1 kanamycin and incubated at 37 °C, 200 rpm until A 600 reached 0.6 before addition of 400 μL of 1 M isopropyl--D-thiogalactopyranoside (IPTG) and incubation at 22 °C overnight to induce protein expression. Cells were harvested by centrifugation at 4000g for 10 min, resuspended in lysis buffer (20 mM Tris-HCl, pH 7.8, 0.5 M NaCl, 10 mM imidazole) and lysed by sonication. The total lysate was centrifuged at 14,000 x g for 40 min, and the supernatant was loaded onto a His-Bind column (1 mL bed volume), which had been precharged with nickel ions and equilibrated with lysis buffer. The column was washed with 10 column volumes of lysis buffer. Bound proteins were then eluted with a step gradient of increasing imidazole concentration (40, 80, 100, 150, 200, 250 and 500 mM in binding buffer). The protein solutions were concentrated, and further purified by gel filtration on an ÄKTA Explorer FPLC system fitted with a HiLoad 16/60 Superdex 200 Prep Grade column.
The mobile phase contained 100 mM potassium phosphate, pH 7.4. Fractions containing protein of the expected size were pooled and concentrated using Amicon Ultra-4 concentrators (Millipore) fitted with either 10 kDa or 30 kDa filter. All purification steps were carried out at 4 °C. The purity of the protein was examined by 4-12% Bis-Tris Gel (Novex) analysis and the concentration of the protein was measured by Bradford assay using bovine serum albumin as a standard.

In vitro activity assays
Amidinohydrolase activity with desulfoclethramycin as substrate Each reaction mixture (50 µL) contained 10 µM purified candidate amidinohydrolase Medi2865 (or Medi4948, or Medi0234), 1 mM purified desulfoclethramycin in 100 mM potassium phosphate buffer pH 7.5. After incubation at 37 °C for 1.5 h, 10 µL of the reaction mixture was taken out, and mixed with 50 µL of methanol. The sample was clarified by

Sulfotransferase activity with mediomycin B as substrate
Mediomycin B was generated in situ using desulfoclethramycin and amidinohydrolase Medi4948. Each reaction mixture (50 µL) contained 10 µM purified Medi4948, 1 mM purified desulfoclethramycin in 100 mM potassium phosphate buffer pH 7.5. After incubation at 37 °C for 1.5 h, 10 L was taken out to check by LC-MS to make sure that desulfoclethramycin was fully converted to mediomycin B. Then purified SMALA_2697 and PAPS cofactor were added to the reaction mixture at a final concentration of 10 µM and 1.5 mM respectively, and the reaction was allowed to continue at 37 °C for 2 h. 10 µL of the reaction mixture was taken out, and mixed with 50 µL of methanol. After centrifugation, the sample was analyzed by HPLC-UV-MS.
Scheme S1: Sulfonated natural products.   130  140  150  160  170 180  GI LDTSALSHVG TRGPL-YGKQ DLEEDEKLGF GIVTSADVMR RGVDEVIDQL   310  320  330  340  350 360 LGQ VEVAPAYDH  - MTRRPDRKDV     with the amidinohydrolase-encoding gene amh_A828 from the marginolactone desertomycin biosynthesis from S. olivaceus Tü4018 [5], showing that this enzyme was not able to convert 1a and 1b to their amino forms.   Putative functions of the encoded proteins were deduced from analyses with the BlastP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The % identity/similarity for the protein in the database with the highest end-to-end similarity is indicated. R designates a gene lying on the opposite strand. The entire genome sequence of S. malaysiensis DSM4137 has been deposited in GenBank where it can be accessed as S. malaysiensis Bioproject PRJNA396489, Biosample SAMN07427119. Putative functions of the encoded proteins were deduced from analyses with the BlastP program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The % identity/similarity for the protein in the database with the highest end-to-end similarity is indicated. The entire genome sequence of S. mediocidicus ATCC23936 has been deposited in GenBank where it has been classified as S. blastmyceticus and can be accessed as S. blastmyceticus Bioproject PRJNA411827, Biosample SAAMN07688521.